Determining effects of environmental conditions on protein folding using single m

描述（由申请人提供）：蛋白质折叠是现代生物学中未解决的主要问题。更好地了解蛋白质折叠机制将有助于阐明为什么一些蛋白质错误折叠，导致阿尔茨海默病，帕金森病，II型糖尿病和一些癌症等疾病（1）。为了了解环境和细胞条件在蛋白质折叠中的作用以及什么条件可能导致错误折叠，重要的是要了解环境因素如温度，溶质和应变的影响。本研究的目的是探讨两种环境因素，力和溶质，对蛋白质折叠的影响。光镊用于探测单个蛋白质分子对沿着特定拉轴的力的响应（2）。溶质可以对涉及大规模构象变化的蛋白质过程产生大而特定的影响（3），因此是光镊实验的天然扰动剂。溶质效应取决于过程中掩埋或暴露的表面的数量和类型，因此可用于探测构象变化。这里概述的实验将探索变性剂尿素和稳定渗透剂甘氨酸甜菜碱（GB）的src SH 3结构域的折叠使用光镊的影响。1.通过绘制GB和尿素的力与溶质浓度的折叠与展开相图，表征变性剂和渗透剂如何影响机械折叠。2.通过使用两种技术确定尿素和GB对src SH 3结构域折叠的影响，探索单分子力实验中的折叠与标准集合实验的不同。溶质效应与用于折叠的<$阿萨有关，<$阿萨中的任何差异都应揭示这些实验之间变性状态集合中结构程度的差异。此外，由于光镊实验是在单个蛋白质分子上进行的，因此它们可以用来观察单个蛋白质的行为如何偏离平均整体行为，从而可能揭示可能导致错误折叠的罕见折叠事件。3.通过确定当使用光镊沿沿着两个不同轴拉动时尿素和GB对src SH 3结构域的折叠或展开速率的影响，表征不同的折叠途径。这些速率与用于折叠到过渡状态和从过渡状态折叠的Δ阿萨有关，因此可以用于表征折叠机制。溶质也可能干扰折叠过程，足以揭示替代折叠途径。

DESCRIPTION (provided by applicant): Protein folding is a major unsolved problem in modern biology. A better understanding of the protein folding mechanism will help elucidate why some proteins misfold, leading to disorders like Alzheimers disease, Parkinson's disease, type II diabetes and some cancers (1). In order to understand the role of the environment and cellular conditions in protein folding and what conditions might lead to misfolding, it is important to understand the effect of environmental factors like temperature, solutes and strain. The goal of this proposal is to explore the effect of two environmental factors, force and solutes, on protein folding using optical tweezers. Optical tweezers are used to probe the response of single protein molecules to force along a specific pulling axis (2). Solutes can have large and specific effects on protein processes involving large-scale conformational change (3) and so are a natural perturbant for optical tweezers experiments. Solute effects depend on the amount and type of surface buried or exposed in a process (¿ASA) and so can be used to probe conformational changes. The experiments outlined here will explore the effects of the denaturant urea and the stabilizing osmolyte glycine betaine (GB) on folding of the src SH3 domain using optical tweezers. 1. Characterize how denaturants and osmolytes affect mechanical folding by developing a folded vs unfolded phase diagram of force vs solute concentration for GB and urea. 2. Explore how folding in single molecule force experiments differs from standard ensemble experiments by determining the effect of urea and GB on folding the src SH3 domain using both techniques. Solute effects are related to ¿ASA for folding and any differences in ¿ASA should reveal differences in the extent of structure in the denatured state ensemble between these experiments. Also, because optical tweezers experiments are performed on single protein molecules, they can be used to look at how behavior of individual proteins deviates from average ensemble behavior, potentially revealing rare folding events that could lead to misfolding. 3. Characterize different folding pathways, by determining the effect of urea and GB on the folding or unfolding rate of the src SH3 domain when pulled along two different axes using optical tweezers. These rates are related to ¿ASA for folding to and from the transition state so can be used to characterize the folding mechanism. Solutes could also potentially perturb the folding process enough to reveal alternate folding pathways.